A plant is an ideal system for producing carbohydrates with low energy load, in which carbohydrates are photosynthetically produced from water and carbon dioxide. Except for a part of organisms, the other organisms can not synthesize the carbohydrates by themselves and utilize the sugars derived from the plants. Meanwhile, each of an animal and a microorganism inherently has the ability to synthesize the carbohydrates by modifying the sugars and extending sugar chains using the sugars derived from the plants as sources, and there are sugar derivatives and carbohydrates derived from the animals and the microorganisms, which the plants can not produce. The carbohydrate which can not be produced in the plants and is produced by the animals and the microorganisms includes hyaluronic acid.
The hyaluronic acid is glycosaminoglycan (mucopolysaccharide) isolated from corpus vitreous body from bovine eye ball by Meyer and Palmer in 1934 (Meyer, K. and Palmer, J. W. (1934) J. Biol. Chem., 107, 629-634). They have demonstrated that this substance is a linear polysaccharide of repeating glucuronic acid β-1,3-N-acetylglucosamine β-1,4 disaccharide units (Weissman, B. and Meyer, K. (1954) J. Am. Chem. Soc., 76, 1753-1757).
Subsequently in 1950s to 1960s, the study on biosynthesis of the hyaluronic acid was performed by a cell-free system from Group A Streptococcus. The streptococcal enzyme activity localized on the membrane fraction was shown by using two sugar nucleotides of uridine-5′-diphosphoglucuronic acid (sometimes referred to as UDP-glucuronic acid or UDP-GlcA) and uridine-5′-diphospho-N-acetylglucosamine (sometimes referred to as UDP-N-acetylglucosamine or UDP-GlcNAc) in production of a hyaluronic acid chain (Markovitz, M., Cifonelli, J. A. and Dorfman, A. (1959) J. Biol. Chem., 234, 2343-2350). It had been difficult for a long time to solubilize and highly purify the hyaluronic acid synthase as a stable active form, but a gene (hasA) encoding the streptococcal hyaluronic acid synthase was cloned in 1993 (DeAngelis, P. L., Papaconstantinou, J. and Weigel, P. H. (1993) J. Biol. Chem., 268, 14568-14571). Since then, clonings of the genes encoding the hyaluronic acid synthase in mammalian cells were reported (Itano, N. and Kimata, K. (1996) J. Biol. Chem., 271, 9875-9878; Itano, N. and Kimata, K. (1996) Biochem. Biophys. Res. Commun., 222, 816-820; Spicer, A. P., Augustine, M. L. and McDonald, J. A (1996) J. Biol. Chem., 271, 23400-23406; Spicer, A. P., Olson, J. S. and McDonald, J. A. (1997) J. Biol. Chem., 272, 8957-8961; Shyjan A. M., Heldin, P., Butcher E. C., Yoshino T. and Briskin, M. J. (1996) J. Biol. Chem., 271, 23395-23399; Watanabe, K. and Yamaguchi, Y. (1996) J. Biol. Chem., 271, 22945-22948), further the genes encoding the hyaluronic acid synthase in chlorella virus PBCV-1 (DeAngelis, P. L., Jing, W. Graves, M. V., Burbank, D. E. and vam Etten, J. L. (1998) Science, 278, 1800-1804) and Passteurella multocida (DeAngelis, P. L., Jing, W. Drake, R. R. and Achyuthan, A. M. (1998) J. Biol. Chem., 273, 8454-8458) have been found, and recombinant enzymes of the active form have been obtained.
Along with advance of these studies, physiological functions of the hyaluronic acid have been widely elucidated, and unique physicochemical properties and biological functions thereof have been demonstrated. High molecular weight hyaluronic acid has been used for the treatment of arthrosis deformans, a surgery aid for ophthalmology, adhesion prevention and acceleration of wound healing. It has been also reported that low molecular weight hyaluronic acid has physiologically active effects, and the application thereof to biomaterials and new medical uses has been anticipated.
Until now, the hyaluronic acid has been produced by extraction from mammalian tissues or microbial fermentation. However, risk of contamination with, for example, transmissible spongiform encephalopathies (prions) or transmission of viruses to humans has been concerned in the extraction from the mammalian tissues. For mammalian cells, maintenance thereof is difficult and requires expensive media, and additionally, a growth rate thereof is slow. Meanwhile for the microbial fermentation, the media containing the sugars and cost for equipment investment are problematic. In Escherichia coli, processing of a protein does not occur, it is likely that inclusion bodies are formed and a product is degraded by protease, which are problematic (Petrides, D. et al. (1995) Biotecnol. Bioeng., 48, 529). When the therapeutic substance is produced in the microorganism, the purification cost becomes extremely expensive in order to prevent endotoxin from contaminating.
From these results, if the source sugars are produced in the plants by photosynthesis and the hyaluronic acid can be produced in the plants using such sugars, it appears to be industrially advantageous in terms of safety and cost.
However, although there are examples in which the proteins derived from the mammalian or microbial cells are expressed in the plants (Giddings, G. et al. (2000) Nat. Biotecnol., 18, 1151-1155; Daniell, H. et al. (2001) Trends Plant Sci., 6, 219-226), conformation and a sugar chain structure required for keeping the function of the protein are different from those in the original organism, and thus, the produced protein often has not had the original function. For example, erythropoietin was expressed in tobacco BY-2 cells, but it had no physiological activity in vivo (Matsumoto, S. et al. (1995) Plant Mol. Biol., 27, 1163-1172).
In conventional technology, proteins derived from the mammalian or microbial cells have been expressed in the plants, and the proteins themselves have been primarily extracted and utilized. It has been scarcely reported that the proteins derived from the mammalian or microbial cells have been expressed in the plants and a substance is produced in the plant by taking advantage of the protein expressed in the plant.
As the substance production in the plants, it has been reported that human β-1,4-galactosyltransferase was expressed in tobacco BY-2 cells and consequently galactose was newly bound by β-1,4 bond to the sugar chain of the glycoprotein conventionally present (Palacpac, N. Q. et al. (1999) Proc. Natl. Acad. Sci. USA, 96, 4692-4697). However, this reaction is the reaction in which one sugar is transferred from one sugar nucleotide using the glycosyltransferase, and is quite different from the reaction in which a macromolecule such as the hyaluronic acid is generated by transferring two types of sugar from types of sugar nucleotides in several thousand times.
The hyaluronic acid synthase has generally multiple transmembrane or membrane-associated domains. When the foreign gene is expressed in the host, in the membrane-bound protein such as hyaluronic acid synthase, the transmembrane or membrane-associated domains can not often keep the correct structure. For example, it has been suggested that in the active hyaluronic acid synthase, one hyaluronic acid synthase protein and about 14 to 18 molecules of cardiolipin which is a one of the common phospholipids present on the membrane form a complex to affect the hyaluronic acid synthase activity (Tlapak-Simmons, V. L. (1999) J. Biol. Chem., 274, 4239-4245). This way, it is predicted that it is considerably difficult to express in the plant the hyaluronic acid synthase which the plant does not naturally have.
Although the technology by which the hyaluronic acid is produced in the plant is anticipated to be highly useful, the hyaluronic acid is the foreign carbohydrate that the plants are incapable of producing, and it has been considered from the conventional technology that it is considerably difficult to produce in the plants the substance which the plants do not produce naturally and to produce the macromolecular substance as the hyaluronic acid.
The present invention primarily intends to express the hyaluronic acid synthase in the plants and produce the hyaluronic acid which is not naturally produced in the plants using plant cells or the plants.